NO129578B - - Google Patents

Description

Method for the production of

éh purified, acid carboxypeptidase.

The present invention relates to a method for producing a purified, acidic carboxypeptidase with a molecular weight of 100,000-122,000, which reacts hydrolytically with the terminal amino acid group with the free carboxyl group in proteins or peptides, exclusively with the formation of amino acids at a pH value of approx. 1.5~ 5.5j preferably 3.0-3.5.

It is known that enzymes of the carboxypeptidase type include carboxypeptidase A and B, which are extracted from the pancreas of animals. ; and C from fruit substances. : Such enzymes as are represented by carboxypeptidase A; and B, are known to react "with the release of amino acids, step by step from the carboxyl group present in proteins or peptides, and the optimum pH value for this reaction is approximately 7.5- The pH range where this, which occurs for carboxypeptidase C, is somewhat on the acidic side with an optimum at around 5.3.

From US patent no. 2,848,371 it is known to produce a<1 >proteinase (endopeptidase) with; one molecular weight of 93,000-98,000 when growing fungi of the genus Aspergillus. From US patent no. of about 93,000-98,000. The acidic carboxypeptidase (exopeptidase) isolated according to the present invention, however, has a molecular weight of 100,000-122,000 and is thus different from the known acidic protease (endopeptidase) with a molecular weight of 93,000-98,000, also what concerns the enzyme itself.

From "Shionogi Kenkyusho Nempo", .13 (1963), . pages..91-98 discussed in "Chemical Abstact", 60 (1964), page 198lg, a carboxypeptidase from Streptomyces sioyaensis is also known. a pH optimum at approx. 8.

In "Nature", 201 (4019), 613 (1964), referenced in "Chemical. Abstract" 60 ( 1964 ), p..10. 978f, a carboxypeptidase C from citrus fruits is described, which is quickly destroyed at pH values in the range -6-10 and below 4.

From "Sei-Papers Inst. Phys. Chem. Res/" (Tokyo), 59 (1965) No. 1, pp. 44-48, especially page 48, referenced in "Chemical Abstacts", 63 (1965), pp. 15,173d , proteases are known which are active at neutral or alkaline pH values.

In "Arch. Biochem. Biophys.", 121 (1967), pp. 555~562 and 128 (1968), pp. 88-94, referenced in "Chemical Abstracts" 67 (1967), pp. 87•852j and 60 (1968) , page 93-095x, a carboxypeptidase with a pH optimum of 6-8,. et , isoelectric. point at 2.2 and a molecular weight of 31 • 300-31. 400,.

Finally it is. in "Proe..' Nat.- Acad. Sei. US", 58 (1967 ), pages I299-I306,. especially page 1300., referenced in "Chemical Abstracts" 67 (.1967 ), pages 113-939q, described a carboxypeptidase C, whose pH optimum is not indicated, and whose enzymatic action is only. determined at the pH value 7.3 - The enzyme is presumably not active in the acidic pH range. t.....

A hitherto unknown type of carboxypeptidase has now been isolated. amino acid released; in the pH range.1.5~5.5, and the specific properties.with respect.to substrate are completely different from those of the known types. This hitherto unknown enzyme is called acid carboxypeptidase in accordance with the enzyme nomenclature given in the "Report of the Commission on Enzymes of the International Union of Biochemistry", published in 196I.

The method according to the invention for the production of

a purified, acidic carboxypeptidase with a molecular weight of 100,000-122,000, which reacts hydrolytically with the terminal amino acid group with the free carboxyl group in proteins or peptides, exclusively., under. formation of amino acids, at a pH value of approx. 1.5-5.5., preferably .. 3.0-3.5, where a fungus of the genus Aspergillus is grown in a manner known per se and a raw product is isolated from the liquid phase of the culture medium, is characterized in that said raw product is subjected to the following purification steps: 1. dissolution in water, absorption on a weak cation exchange resin, elution and subsequent dialysis, ..

2. absorption of the product from step 1 on phosphocellulose, - elution and dialysis, 3. absorption of the product from step 2 on; diethylaminoethyl--: cellulose, elution; and dialysis, and 4. gel filtration on cross-linked dextran, elution, and lyophilization.

In the drawing in fig. 1 shows the diagram of the influence of the pH value on the activity of acid carboxypeptidase. In fig. 2-4 show the results of chromatography of acid carboxypeptidase, using weakly acidic cation exchange resins, e.g. "Duolite CS-101" in fig. 2, phosphorous cellulose in fig. 3 and diethylaminoethyl cellulose in fig. 4. On. fig. 5 shows the results of gel filtration of acid carboxypeptidase using cross-linked dextran of the type commercially available under the trade name "Sephadex G-75".

The optimal pH value for acidic carboxypeptidase according to the invention lies further towards the acidic side than is the case with the known types of carboxypeptidase. For example, the optimum pH value of the enzyme when acting on substrates, such as benzoxycarbonyl-L-tyrosyl-L-leucine, benzoxycarbonyl-L-glutamyl-L-tyrosine and benzoxycarbonyl-glycyl-L-prolyl-L-leucyl-L-glycine respectively3, 5, 3,1

and 3.2. In fig. 1, curve A shows the relationship between the pH value and the enzyme activity when the reaction is carried out in a reaction mixture containing 0.02536 benzoxycarbonyl-L-tyrosyl-L-leucine as substrate,

0.0025$ impure enzyme preparation and 0.005M sodium acetate salt with a temperature of 30°C, and curve B shows the same ratio when 0.025% benzoxycarbonyl-L-glutamyl-L-tyrosine is used as substrate and 0.025% impure enzyme preparation.'

In the following, the substrate specificity for said acid carboxypeptidase is described. For convenience, a terminal carboxylic acid group (hereafter called a C-terminal group) in a substrate-peptide chain will be denoted by R-X-Y, where R denotes amino acids, peptides or other acyl-substituted amino acids or peptides, and X and Y denote L-amino acids . For carboxypeptidases A and B, A will hydrolyze the peptide bond between X and Y when the amino acids in the Y-position of the C-terminal group in a substrate with free carboxyl groups are either neutral or acidic amino acid residues, except for proline, and B shows strong activity in the above cases only when the amino acids in the Y position are basic amino acids. On the other hand, the activity of said acidic carboxypeptidase according to the invention is most strongly influenced by amino acids X. E.g. when the amino acid residue X is an aromatic amino acid residue, such as tyrosine or phenylalanine, the enzyme's hydrolysis activity will be maximal. If X is an amino acid residue such as glutamic acid, the activity is still high, but if X is a leucine residue, the activity will decrease somewhat. Other amino acid residues, such as glycine, valine or proline can be used, but the activity drops considerably.

The hydrolysis activity of said carboxypeptidase on peptides and amides is compared in table I, where the influence of different amino acid residues in the substrate on the activity of the enzyme is shown. The enzyme activity is expressed in Table I by means of the relative amount of enzyme activity which can liberate amino acids from different R-X-Y compounds (with benzoxoxycarbonyl-L-tyrosyl-L-leucine as standard) at 30°C, pH 3.5 and - within one minute , the enzyme being used at the specific concentration having an extinction coefficient of 2.0 at 280 µm, unless otherwise stated.

It is a decisive feature, as will be apparent from the values in Table I, that the carboxyl radical of the substrate must be 'free' for the carboxypeptidase to work. Said enzyme can be identified as a type of carboxypeptidase, as the compound shows no reaction with substrates where the carboxyl radicals are. blocked with amides and the like, e.g. benzoxycarbony 1-L-tyrosyl-L-leucyl-amide , benzoxycarbonyl-L-trypt'o-phanyl-L-phenylalanylamide, benzoxy carbonyl-gly cy I-L-phenylalanyl-amide-, benzoxycarbonyl-L-glycyl-L-leucyl- amide, benzoxycarbonyl-L-alanyl-L-phenylalanyl-amide and the like. However, based on the natural properties shown above and in the following, it is obvious that the enzyme differs from the known carboxypeptidases A and B.

Furthermore, the enzyme differs completely from exopeptidases such as dipeptidase and tripeptidase according to its mode of action. During the enzyme hydrolysis, it is important that the amino radical in the a position in the amino acid residue X, i.e. in a position adjacent to the C end group. Y, must be blocked with acyl radicals or peptides. In other words, said enzyme does not react with dipeptides (e.g. L-tyrosyl-L-leucine, glycyl-glycine, glycyl-L-leucine, glycyl-L-aspartic acid, L-leucyl-glycine), and tripeptides ( for example glycyl-glycyl-glycine, L-alanyl-glycyl-glycine, L-le'ucyl-glycyl-glycine, glycyl-glycyl-L-leucine).

It also appears from table I that hydrolysis with the aforementioned enzyme is most strongly influenced by amino acid residues in the X position, but that amino acid residues in the Y position and in the vicinity of this also cause a certain influence.

The enzyme's hydrolysis rate seems to increase more rapidly when the α-amino radical in the X position is substituted with the benzoxycarbonyl radical than with the acetyl radical. Likewise, the enzyme's substrate specificity is strongly influenced by the amino acid residue adjacent to the C-end group Y, i.e. X, and the enzyme has a weak effect on substrates whose C-eride group is a basic amino acid, e.g. benzoyl-glycyl-L-lysine, qg has a very weak effect on substrates that have proline as the C-end group, e.g. benzoxycarbonyl-L-prolyl-L-Ieucyl-glycyl-L-proline. Other requirements that the enzyme places on the substrate are that the substrate's amino acid residue must be in the L-form. Therefore, the substrate that benzoxycarbonyl-L-tyrosyl-rD-leucine sorn has D-amino acid residues will not result in enzyme activity...- - ■ : The enzyme thus differs completely from both carboxypeptidase A and B in terms of substrate specificity. Other differences the enzyme differs from these peptidases in the following areas: The activity of the acidic carboxypeptidase is not inhibited by metal-complexing reagents such as ethylenediaminetetraacetate (0305M) and o-phenanthroline, which remove metals associated with enzyme-protein by coordinate binding. One can therefore conclude that said enzyme does not contain metal, necessary for catalytic action, and this is a significant difference that distinguishes the enzyme from other carboxypeptidases that contain metals that participate in catalytic reactions.--The known exceptions are carboxypeptidase C which is extracted from citrus fruits, and penicillium peptidase B, which is produced from microorganisms belonging to the genus Penicillium, whose activities are not affected by metal-complexing reagents. However, the difference in these cases is that the enzyme's active pH is considerably lower than for the above two enzymes. Furthermore, the literature mentions that the general properties of penicillium peptidase B are more similar to dipeptidases than to carboxypeptidases.

Unlike normal endopeptidases, the present acid carboxypeptidase will not be able to cleave peptide bonds in proteins and peptides. If you e.g. treats a reaction mixture of said enzyme and protein with trichloroacetic acid to produce a fraction containing high molecular weight protein, the trichloroacetic acid-soluble fraction of the clear filtrate contains only free amino acids, but no peptides, as a decomposition product. The enzyme can thus be clearly distinguished from acidic proteinases or other similar endopeptidases produced by Aspergillus whose optimal pH of action is around 2.5-3.0.

The acid carboxypeptidase present is inactivated at 60°C and higher, and at pH 7.0 and higher.

The enzyme's molecular weight is estimated at approx. 100,000 by "Sephadex" test. The enzyme's molecular weight is determined to be 122,000 according to Yphantis' method. The sedimentation constant for the enzyme at zero concentration is estimated to be 7.3 S. Compared to these values, the molecular weight for acidic protease produced from Aspergillus saitoi is 35,550, and for carboxypeptidases A and B is 34,000 and 34,300 respectively. Moon thus finds yet another difference between the enzyme and the aforementioned other enzymes.

Based on the above experimental values, it can be concluded that the present acid carboxypeptidase is a new type of enzyme that has not existed before. known.. ■■ ." -

The activity of the carboxypeptidase is determined according to the following method: One milliliter of reaction mixture containing a certain amount of acid carboxypeptidase and 5 x 10 benzoxoxycarbonyl 1-L-tyrbix Ir-. L-leucine dissolved in 0-.05M acetate buffer is . (pH 3.5)' or 5 x 10~^M benzoxycarbonyl-L-glutamyl-L-tyrosine in buffer solution of pH 3.1, incubate at 30°C for 20 minutes. At the end of the reaction, the mixture is kept at 30°C for 30 minutes while adding 200 µl of 0.3 M sodium hydroxide solution to inactivate the remaining enzyme. This alkaline reaction mixture is neutralized by adding 200 µl of 2.55 S acetic acid, and then 2 .0 ml of 0.5M citric acid buffer (pH 5.0) and 1 ml of ninhydrin mixture-, which is prepared according to the Yemm-Cocking method (E. Cocking and E.W. Yemm; "Biochem. J"." 58, Vol. XII- (1954) ), and heated in boiling water. After 15 minutes of heating, the mixture is stirred vigorously under ice-cooling and made up to 5 ml by adding aqueous ethyl alcohol (2:1 volume/volume). Finally, the color strength is determined from the ■ ninhydrin^ reagent. by measuring the absorbance' at 570 mu^, which indicates the amount of amino acid..

A unit of enzyme activity is defined as the amount of enzyme which can cleave 1 μM L-leucine from benzoxycarbonyl-L-tyrosyl-L-leucine as substrate at pH 3.5 and 30°C for 1 minute. When other substrates are used, the amount of amino acid cleaved off is expressed as leucine equivalent and as a relative enzyme unit. Leucine - or other amino acids cleaved off - as reaction products can be determined using ninhydrin reagent after development by thin-layer chromatography on silica gel. G

as an adsorbent, or by means of an automatic amino acid analyser.. ■:-■'.

The production of the acidic carboxypeptidase is as follows: Some of the species belonging to the genus Aspergillus and used are: Aspergillus usamii, Aspergillus: saitoi, Aspergillus niger, Aspergillus inuii, Aspergillus aureus, Aspergillus "awamori, Aspergillus nakazawaii, Aspergillus oryzae, Aspergillus sojae, etc. Some examples of strains among these species are: Aspergillus:usamii - R-Q635' (ATC.C 14331), Aspergillussaitoi'R-3813 (A.TCC-r 1^332 ), Aspergillus niger HRRL ,3.30, Aspergillus inuii R.-363I (ATCC 14333), Aspergillus : aureus R-4523, As-ergillus aureus R-6512 (ATCC 14334), Aspergillus awamori R-3523, Aspergillus awamori IAM 2390 (ATCC 14335) , Aspergillus nakazawaii R-6..822Y, Aspergillus, ...ory.zae var., magnasporus A - 1 - 5j Aspergillus sojae KS,,psv. However: .the strains used are not limited to the above-mentioned or variants and mutants of these, but .all.originating among, .species .which .belong to the genus Aspergillus and which can produce acid carboxypeptidase, can be used..

The cultivation of the above species can be carried out in either solid medium or liquid medium...........

When using solid culture (the koji method), media containing wheat bran, defatted soybeans and the like are used, which are mixed with other suitable and necessary reagents, such as ammonium chloride, if necessary, and water. The mixture is sterilized under steam pressure and cooled. After cooling, the culture is inoculated and everything is mixed well. The incubation is carried out at 25-40°C for 50-90 hours.

At the end of the incubation, water is added in an amount of 5-20 times the original volume, and the mixture is set aside for 60-70 minutes, after which the extraction of impure enzyme preparation is carried out under pressure. The extracted liquid is filtered, whereby sus-.; : suspended solids, spores and other foreign substances are removed. The filtrate is cooled to 1-5°C. and 95% ethyl alcohol (2.5" 3.0 times, the starting volume) is added, which is cooled to 1-5°C before addition. The mixture is stirred carefully and set aside for 10 hours or longer. The precipitate is freeze-dried below 0 ,1 mm Hg, and the impure enzyme preparation, containing the acidic carboxypeptidase is thus prepared. Instead of ethyl alcohol, acetone, methanol or isopropanol can be used as precipitation medium.

To carry out submerged culture cultivation, a liquid medium is prepared containing the suitable carbon sources, nitrogen sources and other nutrients needed for the cultivation of the species, and the culture is inoculated. (An example is that an aqueous culture suspension is added to the liquid medium as an inoculum). Wheat bran, starch, glucose and so on are used as the medium's carbon source, and as nitrogen source soybeans, casein, meat extract, etc., are used as organic compounds,

and ammonium chloride and other inorganic substances. Furthermore, ingredients such as phosphates or other 'inorganic salts' can be added. Furthermore, the conditions of the submerged culture are regulated so that the activity of the acidic carboxypeptidase is increased to a maximum by selecting suitable species and other factors. use Aspergillus usamii, and in this case it is advantageous to regulate the medium's pH to between 3.0 and 6.0 and the temperature to around 30°C, after which incubation is carried out for 50-100 hours.

Submerged culture cultivation can be carried out stationary, by shaking, stirring or air circulation. When growing on a large scale, the most effective combination of r^ring-air blowing is used.

After ■ the cultivation has been completed, the filtrate is filtered and the filtrate is concentrated, if necessary, and adjusted to approx. pH 4.0. After another filtration, the crude preparation is precipitated, as with the solid culture, with alcohol, after which the product is freeze-dried to the crude product of acid carboxypeptidase.

The crude enzyme preparation prepared above is purified by: 1. dissolution in water, absorption on a weak cation exchange resin, elution, and subsequent dialysis, 2. absorption of the product from step 1 on phospho-cellulose, elution and dialysis. 3- absorption of the product from step 2 on diethylaminoethyl cellulose, elution, and dialysis, and ■-4.. gel filtration on cross-linked dextran, elution, and lyophilization.

An example of analysis values for a manufactured product is given in Table I below, where the activity for acid carboxypeptidase produced from a solid culture containing wheat bran and with species belonging to the genus Aspergillus is determined.

The production, by. the crude enzyme can be carried out as follows:-. I..840 kg wheat bran dus jes. with ; 500. 1- water. and sterilized in . 40 minutes with. above a vapor pressure of 1.1 kg/cm 2 ..: One is-cooling is inoculated, with 3.kg starting culture of Aspergillus saitoi-R-38l3: which is grown in pure culture separately, and the whole was carefully mixed. The mixture was placed on 1,600 koji trays and incubated for approx. 50 hours at a temperature between 30 and 45°C. The koji trays were turned over once every other day, when the temperature of the culture rose.

When the cultivation of the above medium was finished, the material was transferred to an extractor where the mass was left for 60-70 minutes, and 3,000 1 of water was added. An extraction was carried out under a pressure of 60 kg/cm<2>. One got 2,400 1 liquid, approx. 80% of the added water, which was further filtered by suitable filtration technique, to remove suspended solids, spores, etc. The filtrate was collected in a precipitation tank, where it was cooled to 1-5°C with salt water, and where 2.5-3.0 times the initial volume of 95% alcohol which had previously been cooled to 1-5°C was added. This mixture with alcohol was stirred carefully and left for over 10 hours to complete the precipitation. The precipitate was separated more completely by continuous filtration, and immediately then freeze-dried at 0.1 mm Hg. The dry product was crushed, and 16 kg of crude, acidic carboxypeptidase preparation was collected. The enzyme activity of this crude preparation was 3.14 x 10^ enzyme activity per g, and the yield was 60%. II. A mixture of 90 kg of wheat bran, 160 kg of defatted soybeans and 40 kg of ammonium chloride was sterilized, after adding water, at a pressure of 3 kg/cm^ for 1 hour, and transferred to a 20 m^ fermentation container to which water was added to bring the total volume up to 10 m^.- After adjusting the initial pH to 5.5 and the temperature to 35°C, the mixture was inoculated with spores in suspension, which had previously been purified by careful mixing of 100 g of inoculum culture of Aspergillus saitoi R-3813 with 500 ml of sterile water. The incubation was then carried out under vacuum and stirring at 35°C.

After 4 days of incubation, the culture medium was filtered through suitable filters with filter aid. The filtrate was concentrated under reduced pressure at 35 mm Hg and 35°C. 1500 1 of liquid was obtained, which was adjusted to pH 4.0 with an inorganic syce such as e.g. hydrochloric acid, and filtered again. It was then precipitated in the same way as indicated under I above, using ethyl alcohol, and the crude enzyme was freeze-dried to 20 kg of enzyme in powder form. The acid carboxypeptidase activity was 7.8 c 10^ units per g, and the yield was 60%.

When salting out with ammonium sulphate instead of precipitation

with alcohol which under I and II above gave precipitation with ammonium sulphate at 70% concentration the highest yield of acid carboxypeptidase, i.e.

60%.

The following examples shall illustrate the invention in more detail.

Example 1.

Weakly acidic cation exchanger, "Duolite CS-101", which was pre-buffered with 0.001 M acetate buffer, was loaded onto a chromatography column (2 cm diameter) to a height of 10 cm. 20 ml of solution containing 500 mg of dissolved crude enzyme was added as prepared under II above, and the mixture was chromatographed. The chromatography was carried out using a 300 ml mixing chamber with stirrers which were connected between the crude solution and the chromatography column, and made it possible to operate continuously until the point where the pH of the eluate became the same as the pH of the acetate buffer in the 0.15M starting solution, i.e. pH 5.2. Fractions of 5 ml were then collected.

The result of the chromatography of the acidic carboxypeptidase on "Duolite CS-101" is shown in fig. 2. It was found that while the acidic proteinase fraction (or Aspergillopeptidase A) produced by a species of the genus Aspergillus was almost completely adsorbed on the resin, and therefore eluted only slowly, the enzyme according to the invention was practically not adsorbed and could easily eluted from the resin. There was almost no acid proteinase in the acid carboxypeptidase fraction, which was easily eluted. The yield of the enzyme was around 50-15%, somewhat deviating for each chromatography run. The above-mentioned enzyme fraction was subjected to a further purification step by dialysis against 0.005M acetate buffer (pH 4.0).

"Duolite CS-101" can be replaced by "Amberlite GG-50", with which similar results are obtained.

The second chromatography step consists in working up the acidic carboxypeptidase using phospho-cellulose, which is a cationic ion exchange cellulose. Phospho-cellulose pre-equilibrated with 0.05 M acetate buffer (pH 4.0) was packed into a chromatography column (2 cm diameter), at a height of 50 cm. First, 100 ml of acid carboxypeptidase from the first chromatography step was added, and the chromatography was carried out with 0.05 M sodium chloride dissolved in 0.005 M acetate buffer (pH 4.0) by continuous flow through a 500 ml mixing chamber. Fractions of 5 ml were collected.

The result of the above chromatography on phospho-cellulose is shown in fig. 3. The yield of the enzyme was approx. 60%. The enzyme fractions were collected and dialyzed against 0.05M acetate buffer

(pH 5.0/ and then taken to the next purification step.

The third chromatography step was performed on diethylaminoethyl cellulose which is an anionic ion exchange resin. Diethylaminoethyl cellulose equilibrated with 03005M acetate buffer (pH 5.0) was packed onto a chromatography column (2 cm diameter) to a height of 50 cm. Then 194 ml of acid carboxypeptidase from the previous chromatography step was loaded onto the column, and the chromatography was carried out with sodium chloride dissolved in 0.005M acetate buffer (pH 3.0)

continuously through a 500 ml mixing chamber. 5 ml fractions were collected.

The result of the above chromatography on diethylaminoethyl cellulose is shown in fig. 4. The yield of carboxypeptidase was approx. 60%. The fraction obtained was chromatographed again. The yield was approx. 80%. The active fraction was then collected and dialysed.

After the dialysis in the first, second and third cleaning stages can

the collected fractions are optionally concentrated for storage.

The fourth purification step consisted of gel filtration on "Sephadex G-75". The diameter of the column was 2 cm and 65 cm high. "Sephadex G-75" was equilibrated with 0.01M acetate buffer before loading onto the column. A freeze-dried enzyme preparation prepared above was dissolved in 5 ml of acetic acid solution and poured into the upper part of the column, where the gel filtration was carried out with the same acetic acid solution. Fractions of 5 ml each were collected.

The result of the gel filtration on "Sephadex G-75" is illustrated in fig. 5. The yield was 76%. It was clear from these methods that the present acid carboxypeptidase is eluted much faster than acid proteinase (Aspertillopeptidase A) whose molecular weight is 35,000, which indicates that the enzyme has a higher molecular weight.

The enzyme preparation prepared above was freeze-dried directly or after salting out, to produce pure acid carboxy peptidase preparation.

Furthermore, disk electrophoresis was performed on the above enzyme preparation at pH 8.0 (D.E. Williams, R.A. Reisfeld: Ann. New York Acad. Sei. 121, 373 (1964)), and said enzyme showed that it was homogeneously electrophoretic. The purified enzyme preparation: appears to be homogeneous by ultracentrifugation, and s< dimenterin<g>sconstantei. at zero concentration is calculated to 7.3 S.

Claims (1)

Process for the production of a purified, acidic carboxypeptidase with a molecular weight of 100,000 to 1^2,000, which reacts hydrolytically with the terminal amino acid group with the free carboxyl group in proteins or peptides, exclusively with the formation of amino acids, at a pH value of approx. 1.5 to 5.5, preferably 3.0 to 3.5, where a fungus of the genus Aspergillus is cultivated in a manner known per se and where a crude product is isolated from the liquid phase of the culture medium, characterized in that said crude product is subjected to the following purification steps: 1. dissolution in water, absorption on a weak cation exchange resin, elution and subsequent dialysis, 2. absorption of the product from step 1 on phosphocellulose, elution and dialysis, 3- absorption of the product from step 2 on diethylaminoethyl cellulose, elution and dialysis , and 4. gel filtration on cross-linked dextran, elution and lyophilization.